We have cloned five ATP-gated P2X receptor channels (P2XRs) from the pituitary gland: P2X2R, P2X3R, P2X4R, P2X6R, and P2X7R. Our ongoing work is focused on their structural-functional characterization. When expressed in hypothalamic GT1 cells, activation of the P2X7R induced the rapid opening of an integral ion channel that was permeable to small cations. This was followed by a gradual increase in permeability to fluorescent dyes. Such bi-functional permeation properties of P2X7R could reflect a dilation of integral pore of the channels or integration of another permeation pathway by activated channels, such as pannexins. We provided several lines of evidence indicating that the P2X7R pore dilates: 1. During the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. 2. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. 3. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. 4. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the D18 mutant with deleted P2X7R-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. Together, these results indicate that the P2X7R channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore. We also studied the functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of P2XRs. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was nonfunctional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and &#945;&#946;-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating. In collaboration with the Department of Neurobiology from Johns Hopkins University, we have also worked on interactions between beta-amyloid and P2X4R in neuronal cells. Among others, these experiments revealed that the beta-amyloid fragment 1-42 induced a caspase-3-mediated cleavage of the receptor that slowed channel closure times and prevented agonist-induced internalization of the receptor. Silencing the expression of endogenous P2X4R attenuated the beta-amyloid fragment 1-42-induced neuronal death, while expression of P2X4R in a cell line that does not normally express P2XRs enhanced the toxic effects of this fragment. These findings suggested that beta-amyloid-induced synaptic dysfunction and neuronal death may involve alternations in P2XR trafficking and functions. Our collaborative work with Catholic University in Santiago, Chile, was focused on allosteric modulation of P2X2R by several compounds, mainly acting at receptors ectodomain. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69-residue segment in the C-terminal, termed P2X2b, as donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera, but was absent in oocytes expressing P2X4/2b chimera. Site directed mutagenesis experiments revealed that the Cys-430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide and mitochondrial stress inducers myxothiazol, and rotenone mimicked mercury effects. These experiments revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents, but not P2X2bR and P2X2a-C430A and P2X2a-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the hydrogen peroxide-potentiation. Alkylation of Cys-430 residue with methylmethane-thiosulfonate also abolished the mercury and hydrogen peroxide potentiation. Altogether, these results are consistent with the hypothesis that the Cys-430 residue is an intracellular P2X2aR redox sensor. In collaboration with the Section on Molecular Signal Transduction of NICHD, our group has also contributed to the characterization of STIM1/Orai1-mediated calcium entry. The focus in our work in this collaborative project was on the dependence of I-crac current on phosphoinositides in STIM1/Orai1-expressing cells. We showed that the inhibition of type III PI4 kinase by wortmannin strongly affected the amplitude of the I-crac current. We also show that activation of phospholipase C signaling pathway by angiotensin II receptors caused rapid but incomplete inhibition of I-crac current. These results indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai channel activity.